Due to genome instability, most cancer cells exhibit loss of genetic material containing tumor suppressor genes and collateral loss of other genes. We hypothesize that cancer cells exhibit hemizygous loss of essential genes that uniquely sensitizes them to further suppression of those same genes. To identify cancer cell-specific liabilities that are the result of copy number losses, we performed integrated analyses of genome wide copy number and RNAi profiles and identified 71 genes for which gene suppression specifically inhibited the proliferation of cells possessing partial copy loss of that gene. We identified numerous members of the spliceosome including our top hit SF3B1, a component of the U2 snRNP and member of the U2 sub-complex SF3B. We will test our predictions in breast cancer cell lines, where SF3B1 is hemizygously deleted in ~21% of breast cancers. Aim 1 will test the hypothesis that hemizygous loss of the essential spliceosome gene, SF3B1, sensitizes cells to SF3B1 suppression. We will determine if cell lines with hemizygous deletion of SF3B1 are more susceptible to suppression of SF3B1 than cells lines and non- transformed cells with two copies of SF3B1. We will determine whether SF3B1 expression correlates with copy number loss and if these alterations affect U2 snRNP recruitment and splicing function. We will characterize the mechanism leading to decreased viability of hemizygous SF3B1 cells and determine if these effects can be recapitulated by small molecule inhibitors of SF3B, such as Spliceostatin A. Aim 2 will test the hypothesis that hemizygous loss of SF3B1 results in SF3B1 expression as a limiting factor for U2 snRNP assembly and pre-mRNA splicing. We will use gel filtration chromatography and co-immunoprecipitation to determine how the SF3B complex is assembled and if hemizygous loss of SF3B1 limits the formation of fully assembled SF3B. We will determine if there are more pronounced effects on pre-mRNA splicing in SF3B1 hemizygous cells after SF3B1 knockdown. We will assess pre-mRNA splicing biochemically using transcription-coupled splicing assays and transcriptome-wide using RNA sequencing. This proposal has the potential to create a new approach for cancer treatment that could be extended to a large number of novel therapeutic targets and cancer types.